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PubMed2000

Polymer solutions as a pseudostationary phase for capillary electrochromatographic separation of DNA diastereomers.

The solutions of linear polymers traditionally used for DNA separation have been employed for the capillary electrophoresis (CE) of diastereomers of chemically modified DNA. The selectivity of diastereomeric separation of the phosphorothioate (PS) and 2'-O-methylated (2-OMe) PS oligonucleotides depends on the nature of the polymer additive in the CE background electrolyte. The selectivity of separation for different polymers increases in the line: linear polyacrylamide < polyethylene glycol < polyvinyl pyrrolidone. The separation of oligomer diastereomers was shown to be primarily based on the hydrophobic interaction with the polymer network that acts as a pseudostationary phase. While lowering the temperature resulted in improved separation, the addition of organic modifiers such as formamide, methanol or acetonitrile counteracts the solute adsorption on the polymer network, and decreases the selectivity of DNA diastereoseparation. The effect of molecular mass and concentration of the polymer on the separation selectivity was investigated.

Animals↗
PubMed2000

Recent innovations in the use of charged cyclodextrins in capillary electrophoresis for chiral separations in pharmaceutical analysis.

A review is presented on the use of charged cyclodextrins (CDs) as chiral selectors in capillary electrophoresis (CE) for the separation of analytes in pharmaceutical analysis. An overview is given of theoretical models that have been developed for a better prediction of the enantiomeric resolution and for a better understanding of the separation mechanism. Several types of charged CDs have been used in chiral capillary electrophoretic separation (anionic, cationic, and amphoteric CDs). Especially the anionic CDs seem to be valuable due to the fact that many pharmaceutically interesting compounds can easily be protonated (e.g., amine groups). For that reason several anionic CDs are now commercially available. Cationic and amphoteric CDs are less common in chiral analysis and only a few are commercially available. Attention is paid to the most common synthesis routes and the characterization of the CDs used in chiral capillary electrophoretic separations. The degree of substitution in the synthesized CDs may vary from one manufacturer to another or even from batch to batch, which may have a detrimental effect on the reproducibility and ruggedness of the separation system. In Sections 4, 5, and 6 the applications of anionic, cationic, and amphoteric CDs for the chiral separation in CE are described. Many interesting examples are shown and the influence of important parameters on the enantioselectivity is discussed.

Cyclodextrins↗
PubMed2001

DNA separations in microfabricated devices with automated capillary sample introduction.

A novel method is presented for automated injection of DNA samples into microfabricated separation devices via capillary electrophoresis. A single capillary is used to electrokinetically inject discrete plugs of DNA into an array of separation lanes on a glass chip. A computer-controlled micromanipulator is used to automate this injection process and to repeat injections into five parallel lanes several times over the course of the experiment. After separation, labeled DNA samples are detected by laser-induced fluorescence. Five serial separations of 6-carboxyfluorescein (FAM)-labeled oligonucleotides in five parallel lanes are shown, resulting in the analysis of 25 samples in 25 min. It is estimated that approximately 550 separations of these same oligonucleotides could be performed in one hour by increasing the number of lanes to 37 and optimizing the rate of the manipulator movement. Capillary sample introduction into chips allows parallel separations to be continuously performed in serial, yielding high throughput and minimal need for operator intervention.

Automation↗
PubMed2001

Spatial open-network formed by mixed triblock copolymers as a new medium for double-stranded DNA separation by capillary electrophoresis.

A mixture of two polyoxybutylene-polyoxyethylene-polyoxybutylene (BEB) triblock copolymers (B6E46B6 and B10E271B10, respectively) was used as a new separation medium for separating double-stranded DNA (dsDNA) fragments by capillary electrophoresis (CE). The two block copolymer mixtures were designed to form mixed flower-like micelles in dilute solution and a homogeneous gel-like open-network with hydrophobic clusters as cross-linking points at higher polymer concentrations. Being a polyoxyalkylene block copolymer gel, the separation medium has some special advantages, including the temperature-dependent sol-gel transition that makes sample injection easy, and the self-coating of the inner capillary wall that makes experimental procedures simple and reproducible. Furthermore, it can shorten the elution time and further improve the separation resolution, especially for small dsDNA fragments, when compared with EPE-type separation media, e.g., F127 (E99P69E99, with P being polyoxypropylene) block copolymer gels formed by the closed packing of spherical micelles. Single base pair resolution can be achieved by using the new separation medium for dsDNA fragments up to over 100 base pairs.

Boric Acids↗
PubMed2001

Effects of electrolyte modification and capillary coating on separation of glycoprotein isoforms by capillary electrophoresis.

The capillary electrophoresis (CE) running electrolyte composition was optimized for the separation of selected glycoproteins. A good separation of the ovalbumin (OV) and alpha-acid glycoprotein (AAG) isoforms was achieved in 20 mmol x L(-1) N-(2-hydroxyethyl)piperazine-2'-(2-ethanesulfonic acid) (HEPES) at pH 7.0, in 20 mmol x L(-1) phosphate, pH 7.0, or in 25 mmol x L(-1) borate, pH 7.9. Various ways of suppression of the glycoprotein adsorption onto the capillary wall were compared. Alpha, omega-diamine alkanes and bis(aminoalkyl) amines were added to the CE buffers, the optimized concentration being 1 mmol x L(-1) in 20 mmol x L(-1) phosphate buffer. The OV and AAG isoforms could be separated using all the alpha,omega-diamine alkanes or bis(2-aminoethyl)amine. The length of the alkyl chain in the diaminoalkane did not influence the separation. The separation of the isoforms of pollen allergens was also tested. The effects of modification of the capillary wall by succinyl-poly-L-lysine and hydrophilic CElect-P1 capillary were compared. A decrease in the glycoprotein and protein adsorption resulted in an improved separation of the isoforms.

Buffers↗
PubMed2001

The role of separation science in proteomics research.

In the last few years there has been an increased effort into the separation, quantification and identification of all proteins in a cell or tissue. This is a review of the role gel electrophoresis, high performance liquid chromatography (HPLC), and capillary electrophoresis (CE) play in proteomics research. The capabilities and limitations of each separation technique have been pointed out. Instrumental strategies for the resolution of cell proteins which are based on efficient separation employing either a single high-resolution procedure or a multidimensional approach on-line or off-line, and a mass spectrometer for protein identification have been reviewed. A comparison of the advantages of multi-dimensional separations such as two-dimensional polyacrylamide gel electrophoresis, HPLC-HPLC, and HPLC-CE to the separation of cell and tissue proteins are discussed. Also, a discussion of novel approaches to protein concentration, separation, detection, and quantification is given.

Chromatography, High Pressure Liquid↗
PubMed2001

Separation of double-stranded DNA fragments by capillary electrophoresis in interpenetrating networks of polyacrylamide and polyvinylpyrrolidone.

Mixtures of two polymers with totally different chemical structures, polyacrylamide and polyvinylpyrrolidone (PVP) have been successfully used for double-stranded DNA separation. By polymerization of acrylamide in a matrix of PVP solution, the incompatibility of these two polymers was suppressed. Laser light scattering (LLS) studies showed that highly entangled interpenetrating networks were formed in the solution. Further systematic investigation showed that double-stranded DNA separation was very good in these interpenetrating networks. With a concentration combination of as low as 2% w/v PVP (weight-average molecular mass Mr = 1 x 10(6) g/mol) + 1% w/v polyacrylamide (Mr = 4 x 10(5) g/mol), the 22 fragments in pBR322/HaeIII DNA, including the doublet of 123/124 bp, have been successfully separated within 6.5 min. Under the same separation conditions, similar resolution could only be achieved by using polyacrylamide (Mr = 4 x 10(5) g/mol) with concentrations higher than 6% w/v and could not be achieved by using only PVP (Mr = 1 x 10(6) g/mol) with a concentration as high as 15% w/v. It is noted that the interpenetrating network formed by 2% PVP and 1% polyacrylamide has a very low viscosity and can dynamically coat the inner wall of a fused-silica capillary. The separation reached an efficiency of more than 10(7) theoretical plate numbers/m and a reproducibility of less than 1% relative standard deviation of migration time in a total of seven runs. The interpenetrating network could stabilize polymer chain entanglements. Consequently, the separation speed was increased while retaining resolution.

Acrylic Resins↗
PubMed2002

Predicting and evaluating separation quality of micellar electrokinetic capillary chromatography by artificial neural networks.

Computer-aided optimization of micellar electrokinetic capillary chromatography (MEKC) separations was demonstrated by artificial neural networks (ANNs) using a Levenberg-Marquardt algorithm and an orthogonal experimental design. A novel criterion, named Q, for evaluating the separation quality of MEKC was firstly presented, which considered both separation selectivity and analysis time. MEKC separation conditions of seven plant hormones were then simulated and optimized using ANNs based on this novel criterion. The result was further compared to that obtained using ANNs based on a traditionally used criterion of overall normalization resolution (named r). Finally, the separation under optimum conditions predicted by ANNs using the criterion Q was compared to, and proved to be better than that obtained by empirical step-by-step optimization procedures. This method may also be adapted to other separation methods due to its generality.

Algorithms↗
PubMed2002

On-line coupling of capillary isoelectric focusing with transient isotachophoresis-zone electrophoresis: a two-dimensional separation system for proteomics.

A microdialysis junction is employed as the interface for on-line coupling of capillary isoelectric focusing with transient isotachophoresis-zone electrophoresis in a two-dimensional separation system. Capillary isoelectric focusing not only provides high-resolution separation of tryptic peptides based on their differences in isoelectric point, but also potentially allows the analysis of low-abundance proteins with a typical concentration factor of 50-100 times. Carrier ampholytes, employed for the creation of a pH gradient during focusing, are further utilized as the leading electrolyte in the second separation dimension, transient isotachophoresis-zone electrophoresis. Many peptides which have the same isoelectric point would most likely have different charge-to-mass ratios, and thus different electrophoretic mobilities in zone electrophoresis. Two-dimensional separation of proteolytic peptides is demonstrated using standard proteins, including cytochrome c, ribonuclease A, and carbonic anhydrase II. The maximum peak capacity is estimated to be around approximately 1600 and can be significantly increased by simply increasing the capillary column length and manipulating the range of pH gradient in isoelectric focusing. In addition to enhanced separation efficiency and resolution, this two-dimensional electrokinetic separation system permits sensitive and comprehensive analysis of peptide fragments, especially when integrated with electrospray ionization mass spectrometry for peptide/protein identification.

Amino Acid Sequence↗
PubMed2002

Fast choice of separation conditions for analyses by capillary zone electrophoresis using an information system Xemic.

An information system Xemic applicable in analytical chemistry is described and its use in capillary electrophoresis for searching suitable separation conditions is demonstrated. This system is capable to provide suitable separation conditions even for analytes for which no electrophoretic experiments have been published so far. The system works with a database of components of anionic character the analyses of which have been performed, published in reviewed scientific journals, and included into a database created by an expert. Moreover, the system learned to search also in abstracts or complete scientific articles to find articles dealing with the determination of a substance in a given sample matrix. When no experiments have been published so far for a defined substance in a specific matrix, Xemic shows the separation conditions for determination of the substance regardless of the matrix. When no response can be found for the analyte of interest at all, the system Xemic works like an expert in the field and searches chemically similar substances and offers a series of substances the physicochemical properties of which are close to the followed analyte with respect to the behavior in the electric field, and shows working conditions for their analysis. Thus, the analyst puts only the order in the form of a given analyte in a given matrix and obtains a recommendation of a separation system that should enable to perform a successful separation. The system is not rigid and enables the operator to change the importance of individual attributes used in similarity search so as to obtain a broader or narrower group of similar components. With a certain probability the analyte of interest can be successfully analyzed under separation conditions that suited for the analysis of the most similar substances in the given matrix.

Computers↗
PubMed2002

A miniaturized multichamber solution isoelectric focusing device for separation of protein digests.

A miniaturized multichamber device was constructed for solution isoelectric focusing (IEF) separation of complex peptide mixtures. The system, based on immobilized pH gels, consisted of 96 minichambers ( approximately 75 nuL each) arranged in eight rows. Neighboring chambers in a given row were separated by short glass tubes (4 mm inner diameter, 3 mm long), within which Immobiline gels of specific pH values were polymerized. During focusing, the device was sandwiched between two supporting blocks incorporating the reservoirs for anolyte and catholyte. In principle, multiple samples could be simultaneously fractionated, each separated into 12 fractions of various pI ranges. A variety of standard peptide mixtures and tryptic digests of proteins were separated by IEF using this device, and the fractions were characterized by mass spectrometry. For a codigested nine-protein mixture, both the total number of peptides identified and the average sequence coverage were similar to the results of ion-exchange chromatography (IEC), according to matrix assisted laser/desorption/ionization--time of flight (MALDI-TOF) data. The IEF separation provided concentrated and desalted fractions, suitable for an additional separation liquid chromatography, capillary electrophoresis (LC, CE) or mass spectrometry (MS) detection without additional sample cleanup. High loading capacity was achieved for the miniaturized multichamber IEF device. Importantly, a linear correlation was found between the experimentally determined and calculated pI values of peptides.

Acrylamides↗
PubMed2002

DNA separation by microchip electrophoresis using low-viscosity hydroxypropylmethylcellulose-50 solutions enhanced by polyhydroxy compounds.

Low-viscosity polymer solutions have potential for double-stranded (ds) DNA separations in micrototal analysis systems (micro-TAS). In this paper, we report dilute, low-viscosity hydroxypropylmethylcellulose-50 (HPMC-50, 11.5 kDa) solutions containing polyhydroxy additives as separation media. Predominant operational variables, such as applied electric field strength, fluorescent intercalator (YOPro-1) concentration, polymer concentration, and additive concentration, are thoroughly investigated. Fast (within 170 s) and excellent separation of DNA restriction fragments ranging in size from 72 to 1353 base pairs (bp) is achieved in a 30 mm length channel of polymethylmethacrylate (PMMA) microchips at an electric field strength of 300 V/cm, by introducing 8% mannitol, 8% glucose or 10% glycerol additives into a 2% HPMC-50/1 x Tris-borate-EDTA (TBE) solution. The low-viscosity (40 cP) matrix formulation provides both coating of the microchannels and separation of DNA in one step. The performance in the solution surpasses that in highly concentrated HPMC-50 solution. In addition, separation using 1xTris-EDTA buffer in the 2% HPMC-50 matrix containing polyhydroxy additives also exhibits a notably increased performance. This is presumably due to formation of hydrogen-bonding interactions of polyhydroxy additives with HPMC-50 matrix and DNA so as to increase the coupling interactions between matrix and DNA molecules during electrophoresis. The result reflects that boric acid is not a prerequisite in polyhydroxy-enhanced HPMC-50 solution for separation.

Bacteriophage phi X 174↗
PubMed2005

Separation of peroxidation products of diacyl-phosphatidylcholines by reversed-phase liquid chromatography-mass spectrometry.

Lipid peroxidation process has attracted much attention due to the growing evidence of its involvement in the pathogenesis of age-related diseases. The monitoring of the lipid peroxidation products in phospholipids, formed under oxidative stress conditions, may provide new markers for oxidative stress signaling and for disease states, giving new insights in the pathogenesis process. Reversed-phase liquid chromatographic method coupled to mass spectrometry was developed for the separation of oxidized glycero-phosphatidylcholine (GPC) peroxidation products formed by the Fenton reaction that mimic in vivo oxidative stress conditions. The LC-MS conditions were applied for the separation of peroxidation products of oleoyl- (POPC), lineloyl- (PLPC) and arachidonoyl-palmitoyl phosphatidylcholine (PAPC). The peroxidation products separated included products resulting from the insertion of oxygen atoms in the sn-2 chain (long-chain), and products with the sn-2 chain shortened resulting from cleavage of oxygen-centered radicals (short-chain). Among long-chain products were the keto, hydroxy, hydroperoxide and poly-hydroxy derivatives, while short-chain products included dicarboxylic acids, aldehydes and hydroxy-aldehydes. Separation of long-chain products formed in each phosphatidylcholine was observed, and the reconstructed ion chromatogram of each ion showed an increase in the number of peaks with the increase in the number of oxygen atoms inserted into the phospholipid. Separation of short-chain products took place according to the functional group present at the sn-2 moiety that allowed the elution of dicarboxylic acids distinct from aldehydes. Separation between isomeric structures that were present in short- and long-chain products was also achieved.

Chromatography, High Pressure Liquid↗
PubMed2001

Separation of naturally occurring triterpenoidal saponins by capillary zone electrophoresis.

A high-performance capillary electrophoresis (HPCE) was successfully applied to the separation and quantitation of naturally occurring oleanene triterpenoidal saponins. The HPCE adapted to the separation of two pairs of disteriomeric saponins (1-2) or (3-4), obtained from Trifolium alexandrinum seeds, was based on capillary zone electrophoresis (CZE) in borate buffer with UV detection at 195 nm. An usual technique for isolation and group separation of saponins was developed as an appropriate purification step prior to determination of individual saponins by CZE. The separation parameters such as borate concentration, pH and applied voltage were varied in order to find the best compromise that complied with demands for high separation, short duration and sufficiently high detector response. The optimum running conditions were found to be 60 mM borate buffer, pH 10 and 12 kV. Under the alkaline borate electrolyte, no resolution was achieved for the saponins (1 and 3) or (2 and 4) in a single mixture, except when 20 mM beta-cyclodextrin was added to the running electrolyte. With the combined techniques of group separation, purification and CZE, a rapid and efficient method for the determination of naturally occurring diasteriomeric saponins is now available.

Buffers↗
PubMed2003

Phase separation in metal oxides.

A fascinating phenomenon, recently found to occur in certain transition-metal oxides, is phase separation wherein pure, nominally monophasic oxides of transition metals with well-defined compositions separate into two or more phases over a specific temperature range. Such phase separation is entirely reversible, and is generally the result of a competition between charge-localization and -delocalization, the two situations being associated with contrasting electronic and magnetic properties. Coexistence of more than one phase, therefore, gives rise to electronic inhomogeneity and a diverse variety of magnetic, transport, and other properties, not normally expected of the nominal monophasic composition. An interesting feature of phase separation is that it covers a wide range of length scales anywhere between 1-200 nm. While cuprates and manganates, especially the latter, provide excellent examples of phase separation, it is possible that many other transition-metal compounds with extended structures will be found to exhibit phase separation.

Journal Article↗
PubMed2003

Comprehensive strategy for chiral separations using sulfated cyclodextrins in capillary electrophoresis.

This review focuses on the emerging role of sulfated cyclodextrins in the capillary electrophoretic (CE) separation of chiral analytes. Since being introduced as enantioselective agents for CE in 1995, these anionic additives have continued to demonstrate remarkable application universality. The broad spectrum of chiral compounds successfully separated using this approach includes acidic, basic, neutral, and zwitterionic species. This impressive array of analyte structures is derived from a growing diversity of compound classes including pharmaceuticals, plant extracts, biomarkers, herbicides, alkaloids, fungicides, and metal ions. Moreover, literature reports highlight the minimal optimization required to achieve a successful separation. Based on these findings, sulfated cyclodextrins appear to be well suited for the development of a more universal, comprehensive separation strategy for chiral compounds. This review explores this proposition by beginning with the structure and migration properties of sulfated cyclodextrins, using applications to highlight the separating power of this technique and ending with a pragmatic, comprehensive separation strategy.

Animals↗
PubMed2002

Effects of neonatal handling and maternal separation on rough-and-tumble play in the rat.

The extent to which brief daily handling and longer periods of separation from the mother during the first 2 weeks of life can affect play behavior in juvenile rats was assessed. Rat pups were separated from the mother for either 15 min daily (handling) or for 3 hr daily (maternal separation), and play was observed as juveniles. Overall levels of playfulness were not affected by either manipulation, although certain aspects of playful responsiveness were affected in males, but not females. In particular, the pattern of responsiveness to playful contacts was feminized in both handled and separated male rats. Activity in a novel open field at 15 days of age was increased in both males and females from the separated group, but not in the handled animals, as were the number of rears exhibited during the play bouts. These data suggest that early rearing experiences can have subtle gender-dependent effects on some aspects of play in juvenile rats and that the underlying mechanism(s) responsible for these effects may differ from those associated with other effects reported for handling and maternal separation.

Animals↗
PubMed1989

Behavioral and autonomic responses to peer separation in pigtail macaque monkey infants.

Brief maternal separations of young nonhuman primates have been used extensively to study the behavior and physiology of attachment, loss, and bereavement. The physiological responses to the loss of alternative attachment figures, such as peers, is less well documented in nonhuman primates. This study examined both autonomic and behavioral responses of peer-reared pigtail macaque infants to separation. Eight infants were removed from their mothers at birth and reared in four peer pairs. At 6 months of age, each monkey was implanted with a multichannel biotelemetry device which transmitted heartrate, body temperature, EEG, EMG, and EOG. Blood was collected twice weekly for immunological assessment. Behavioral and physiological data, including sleep, were collected for 1 week of baseline, 2 weeks of separation, and 1 week of reunion. Behavioral and physiological results indicated agitation but not depression following separation from their peer attachment figures. We found reduced mitogenic responses to pokeweed consequent to peer separation, suggestive of altered B-cell function. REM variables were the only sleep measures affected by the separation, and were suggestive of agitation but not depression.

Animals↗